The present invention relates to methods of treating immunity-related diseases via modulation of differentiation of immunostimulatory/mature antigen-presenting cells, and further relates to articles of manufacture for practicing such treatment methods. More particularly, the present invention relates to methods of treating diseases characterized by pathological or insufficient immune responses, via modulation of thrombospondin-mediated endocytosis of apoptotic cells by antigen-presenting cells, and further particularly relates to the use of agonists or inhibitors of interactions between thrombospondin and thrombospondin receptors for practicing such disease treatment methods, respectively.
Immunity-related diseases comprise a large number of diseases characterized by significant mortality and morbidity, and for which no satisfactory/optimal treatments are presently available. Such diseases include those characterized by pathological immune responses, such as autoimmune, transplantation-related, inflammatory and alloimmune pregnancy diseases; and those characterized by insufficient immune responses, such as infectious and/or tumoral diseases.
Antigen-presenting cells, such as dendritic cells, play pivotal roles in modulation of immune responses. Under non-inflammatory conditions, immature dendritic cells in peripheral tissues continuously capture innocuous and cell-associated self-antigens and migrate to draining lymph nodes, where they can induce tolerance to such antigens (Steinman, R. M. et al., 2003. Tolerogenic dendritic cells. Annu. Rev. Immunol. 21:685-711). In contrast, under pathological conditions, such as in the presence of pathogens and necrotic cells, dendritic cells undergo a process of maturation involving up-regulation of costimulatory molecules, secretion of proinflammatory cytokines, and acquisition of the capacity stimulate the differentiation of naive T-cells into effector cells. The process of endocytosis of apoptotic cells by antigen-presenting cells, such as dendritic cells and macrophages, has been suggested to play an important role in maintenance of immune homeostasis, via resolution of inflammation, and via induction of peripheral immune tolerance (Savill, 2001; Vandivier et al., 2002; Verbovetski et al., 2002). It has been suggested that apoptotic cells facilitate their endocytosis by generating pro-endocytotic signals to professional phagocytes, and antigen-presenting cells, and that such endocytosis occurs in the absence of inflammatory or autoimmune responses (Voll et al., 1997; Fadok et al., 1998; Huynh et al., 2002; Verbovetski et al., 2002; Savill et al., 2002). Direct pro-endocytotic signals generated by apoptotic cells include alterations in cell surface phospholipid composition (Fadok et al., 1992), changes in cell surface glycoprotein expression, or distinct alterations in cell surface charge (Henson et al., 2001). As well, certain serum proteins can opsonize an apoptotic cell surface and signal phagocytes to endocytose the opsonized apoptotic cells (Mevorach et al., 1998; Mevorach, 1999; Verbovetski et al., 2002), and apoptotic cells can secrete molecules, such as lysophosphatidylcholine, so as to attract phagocytes (Lauber et al., 2003). In contrast to apoptotic cells, viable cells provide signals actively preventing their endocytosis, for example via restriction of phosphatidylserine to the inner leaflet of their membrane, and by surface display of CD31, which is down-regulated upon apoptosis (Brown et al., 2002). The range of mechanisms involved in mediating and regulating identification and clearance of apoptotic cells indicate that such processes are essential for proper maintenance of immune homeostasis.
Thrombospondins are a family of extracellular glycoproteins consisting of five members in vertebrates: thrombospondin (TSP)-1 (TSP-1), thrombospondin-2, thrombospondin-3, thrombospondin-4, and thrombospondin-5/cartilage oligomeric matrix protein. Thrombospondin-1, which is secreted by macrophages and dendritic cells (Savill et al., 1992, Doyen et al., 2003), fibroblasts (Moodley et al., 2003) and other cell types (Adams, 2001), has been implicated in mediating endocytosis of apoptotic cells by antigen-presenting cells (Savill et al., 1992; Moodley et al., 2003; Stern et al., 1996). This molecule is a homotrimeric glycoprotein composed of subunits each having a molecular weight of approximately 145 kilodalton, which was first described as a platelet alpha-granule protein that is released upon activation (Baenziger et al., 1971), and which has been found to mediate numerous cell-matrix and cell-cell activities through a variety of receptors (reviewed by Adams, 2001). Thrombospondin-1 has an N-terminal, heparin-binding domain (HBD) which is cleaved and released upon platelet aggregation (reviewed in Elzie et al., 2004), and which is capable of specifically binding CD29/beta1 integrin, as evidenced by its capacity to specifically bind at least three different beta1 integrins, including alpha3beta1, alpha6beta1, and alpha4beta1 integrins, (Krutzsch, H. C. et al., 1999. J. Biol. Chem. 274:24080-24086; Chandrasekaran, L. et al., 2000. Mol. Biol. Cell 11, 2885-2900; Calzada, M. J. et al., 2003. J. Biol. Chem. 278:40679-40687). The heparin-binding domain has been suggested to mediate thrombospondin-1-induced angiogenesis (Chandrasekaran, L. et al., 2000. Mol. Biol. Cell 11, 2885-2900), cell adhesion, and cellular chemotaxis (Krutzsch, H. C. et al., 1999. J. Biol. Chem. 274:24080-24086; Calzada, M. J. et al., 2003. J. Biol. Chem. 278:40679-40687), but has not been implicated in regulation of immunostimulatory differentiation/maturation of antigen-presenting cells.
In view of the role of thrombospondin-1 in mediating endocytosis of apoptotic cells by antigen-presenting cells, and in view of the role of such endocytosis in inhibition of differentiation of immunostimulatory/mature antigen-presenting cells a potentially advantageous strategy for treating immunity-related diseases may be to suitably modulate thrombospondin-1-induction of such differentiation.
Various approaches have been proposed in the art for modulating thrombospondin-1-mediated inhibition of differentiation of immunostimulatory/mature antigen-presenting cells.
One approach which has been suggested for stimulating such differentiation involves using thrombospondin-1 at agonistic concentrations of 0.4 to 10 micrograms per milliliter, in an attempt to increase endocytosis of apoptotic neutrophils by macrophages (Savill et al., 1992).
Several prior art approaches have been suggested, as follows, for decreasing/eliminating thrombospondin-1-mediated inhibition of differentiation of immunostimulatory/mature antigen-presenting cells.
One approach involves using soluble thrombospondin-1 at inhibitory/blocking concentrations. This approach has been attempted using thrombospondin-1 at blocking concentrations of 100 micrograms per milliliter, in an attempt to inhibit endocytosis of apoptotic neutrophils by macrophages (Savill et al., 1992).
Another approach involves using anti-thrombospondin-1 antibodies for blocking interactions of thrombospondin-1 with receptors thereof. This approach has been attempted using a monoclonal antibody (A6.1) specific for the EGF repeat motif of thrombospondin-1, in an attempt to inhibit endocytosis of apoptotic neutrophils by macrophages (Savill et al., 1992); a monoclonal antibody (A2.5) specific for the N-terminal domain of thrombospondin-1, in an attempt to inhibit endocytosis of apoptotic neutrophils by macrophages (Savill et al., 1992); or anti-thrombospondin-1 antibody, in an attempt to inhibit endocytosis of apoptotic eosinophils by antigen-presenting cells (Stern et al., 1996).
Yet another approach involves disrupting interactions between thrombospondin-1 and the thrombospondin-1 receptor CD36, a ligand of the internal/non-N-terminal type 1 repeats of thrombospondin-1. This approach has been attempted using blocking antibodies specific for CD36, in an attempt to inhibit endocytosis of apoptotic neutrophils (Savill et al., 1992), eosinophils (Stern et al., 1996) or fibroblasts (Moodley et al., 2003) by macrophages; administering antibodies specific for CD36, in an attempt to inhibit LPS-induced dendritic cell maturation, as determined via inhibition of T-cell activation and pro-inflammatory cytokine production (Urban B C. et al., 2001. Proc. Natl. Acad. Sci. U.S.A. 98:8750-8755); or by administering a monoclonal antibody (A4.1) specific for the CD36-binding central stalk-like region of thrombospondin-1 (type 1 repeats), in an attempt to inhibit endocytosis of apoptotic fibroblasts by macrophages (Moodley et al., 2003).
Still another approach involves disrupting the interaction between thrombospondin-1 and CD47, a thrombospondin-1 receptor binding the C-terminal portion of thrombospondin-1. This approach has been tried using blocking antibodies specific for CD47, in an attempt to inhibit bacteria-induced dendritic cell maturation, as determined via inhibition of pro-inflammatory cytokine production (Demeure C E. et al., 2000. J Immunol. 164:2193; Doyen et al., 2003); or using a monoclonal antibody (C6.7) specific for the extreme C-terminus of thrombospondin-1, in an attempt to inhibit endocytosis of apoptotic neutrophils by macrophages (Savill et al., 1992), or immunosuppressive cytokine production by dendritic cells (Doyen et al., 2003).
A further approach involves disrupting specific interactions between C-terminal RGD repeats of thrombospondin-1 and alphaVbeta3 integrin. This approach has been attempted using blocking antibodies specific for CD51/alphaV integrin, in an attempt to inhibit LPS-induced dendritic cell maturation, as determined via inhibition of T-cell activation and pro-inflammatory cytokine production (Urban B C. et al., 2001. Proc. Natl. Acad. Sci. U.S.A. 98:8750-8755); by administering a monoclonal antibody specific for integrin alphaVbeta3, a potential thrombospondin-1 receptor, in an attempt to inhibit endocytosis of apoptotic eosinophils (Stern et al., 1996) or apoptotic fibroblasts (Moodley et al., 2003) by macrophages; or by inducing down-regulation of surface-display of alphaVbeta3 integrin in macrophages, in an attempt to inhibit endocytosis of apoptotic neutrophils (Savill et al., 1992).
An additional approach involves exposing antigen-presenting cells to compounds, such as RGDS peptide or heparin, capable of binding the heparin-binding domain (Beppu R. et al., 2001. Immunol Invest. 30:143-56).
However, the prior art approaches for modulating thrombospondin-1-mediated inhibition of differentiation of immunostimulatory/mature dendritic cells suffer from various critical drawbacks, including: not having been tested in-vivo; not having been investigated using dendritic cells, the antigen-presenting cell type having the most potent immunomodulatory capacity; resulting in induction of both immunostimulatory and immunosuppressive differentiation; being ineffective or suboptimally effective; and/or, having failed to demonstrate any capacity to modulate differentiation of immunostimulatory/mature antigen-presenting cells, such as with respect to surface expression of costimulatory molecules.
Critically, no prior art approach has demonstrated any therapeutic capacity.
Thus, all prior art approaches have failed to provide an adequate solution for treating immunity-related diseases by suitably modulating thrombospondin-1-mediated inhibition of differentiation of immunostimulatory/mature antigen-presenting cells.
There is thus a widely recognized need for, and it would be highly advantageous to have, novel and effective methods ands medicaments for treating immunity-related diseases devoid of the above limitation.